Phosphatidic acid phosphatase (PAP) (also referred to in the art as phosphatidate phosphohydrolase) is known to be an important enzyme for glycerolipid biosynthesis. In particular, PAP catalyzes the conversion of phosphatidic acid (PA) (also referred to in the art as phosphatidate) into diacylglycerol (DAG). DAG is an important branch point intermediate just downstream of PA in the pathways for biosynthesis of glycerophosphate-based phospholipids (Kent, Anal. Rev. Biochem. 64: 315–343, 1995).
In eukaryotic cells, PA, the precursor molecule for all glycerophospholipids, is converted either to CDP-diacylglycerol (CDP-DAG) by CDP-DAG synthase (CDS) or to DAG by phosphatidic acid phosphatase (PAP). In mammalian cells, CDP-DAG is the precursor to phosphatidylinositol (PI), phosphatidylglycerol (PG), and cardiolipin (CL); whereas diacylglycerol is the precursor to triacylglycerol (TG), phosphatidylethanolamine (PE), and phosphatidylcholine (PC) in all eukaryotic cells. Therefore, the partitioning of phosphatidic acid between CDP-diacylglycerol and diacylglycerol is an important regulatory point in eukaryotic phospholipid metabolism (Shen et al., J. Biol. Chem. 271: 789–795, 1996).
In addition to being an important enzyme for glycerolipid biosynthesis, PAP is also an important enzyme for signal transduction. PAP catalyses the dephosphorylation of PA to DAG. DAG is a well-studied lipid second messenger which is essential for the activation of protein kinase C (Kent, Anal. Rev.Biochem. 64: 315–343, 1995); whereas PA itself is also a lipid messenger implicated in various signaling pathways such as NADPH oxidase activation and calcium mobilization (English, Cell Signal. 8: 341–347, 1996). The regulation of PAP activity can therefore affect the balance of divergent signaling processes that the cell receives in terms of PA and DAG (Brindley et al., Chem. Phys. Lipids 80: 45–57, 1996).
Various forms of PAP have been isolated in porcine (Kai et al., J. Biol. Chem. 271: 18931–18938, 1996) and rat species (Brindley et al., Chem. Phys. Lipids 80: 45–57, 1996). Furthermore, the putative amino acid sequence of murine PAP has been identified. Kai et al., supra. Prior to the instant invention, however, human PAP had not been identified or isolated.
Genes coding for PAP have been identified in E. coli (Dillon et al, J. Biol. Chem. 260: 12078–12083, 1985) and in mouse (Kai et al., J. Biol. Chem. 271: 18931–18938, 1996). Furthermore, the following GenBank human cDNA clones are available: accession nos. H17855, N75714, and W70040. No uses were known, however, for these polynucleotide sequences.
Accordingly, there is a need for the identification and isolation of human PAP and for methods of using human PAP, for example, for the dephosphorylation of a substrate.